The Research objective of this proposal is to test whether brain site-specific gene transduction with the use of adeno-associated virus (AAV) can focally restore expression and functions of a disrupted gene in knockout mouse brains. We have been investigating the differential roles of two types of estrogen receptors (ERs), alpha, and beta, in estrogenic regulation of neuroendocrine and behavioral functions, using knockout mice for ER-alpha (alphaRKO) and ER-beta (betaERKO) genes. Knockout mice are great tools to study specific gene function in many traits and correlate them all, but have the limitation that targeted genes are disrupted globally and permanently. Here, we propose to re-introduce a disrupted gene to knockout mice in a specific brain region depending on the endpoint of specific studies. Our preliminary studies demonstrated that we could site-specifically re-introduce a disrupted gene, ER-alpha or ER-beta, to respective knockout mouse brains. It was also found that transduced ER gene was functional, since progesterone receptor protein, the most well characterized down stream product of ligand-dependent transcriptional activity of ER, was induced by estrogen in the transduced cells. In the proposed studies, we will test the functional consequences of site-specific AAV mediated gene transfer in adult knockout mouse brains. Specifically, we will re-introduce ER-6 gene by injecting AAV vectors containing ER-beta Cdna (AAV.ER-beta) into the hypothalamic paraventricular nucleus of betaERKO male mice and determine whether disrupted neuroendocine and behavioral functions can be restored. In the first part of the proposed studies, the effects on estrogen-inducible up-regulation of oxytocin gene expression will be examined with in situ hybridization and quantitative PCR assays (Aim I). Given a positive outcome, behavioral effects of ER-13 gene transduction will be further examined in terms of ER-beta mediated anxiolytic action of estrogen as well as estrogen-inducible aggression in male betaERKO mice (Aim II). The outcome of the proposed studies will provide valuable information about mechanisms of action of two types of ERs in the brain.